Abstract
A yolk protein, egg-specific protein, synthesized and accumulated in the developing ovaries of Bombyx mori serves not only as the nutritive source for embryogenesis but also for the reorganization of the yolk system through limited degradation. Using the purified egg-specific protein as a substrate, a protease responsible for its limited hydrolysis was identified in embryonating eggs and purified to homogeneity. The protease had an apparent molecular mass of 30,500 with one subunit of 29,000 daltons. It hydrolyzes synthetic substrates at carbonyl bonds of Arg or Lys residues, and the hydrolysis is strongly inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and leupeptin, suggesting that it is a trypsin-like protease. The protease shows an extremely high degree (over 2,000-fold) of specificity for egg-specific protein compared to other yolk proteins. Intact egg-specific protein is cleaved into three fragments in two steps; the first releases a 8.7-kDa peptide as an end product and a 55-kDa peptide intermediate, and in the second the intermediate is cleaved into 36- and 17.2-kDa peptides. By relating the NH2-terminal amino acid sequences of these peptides to the sequence of the intact egg-specific protein, the protease was shown to cleave first at a Lys-Asn site and secondly at Arg-Asp. Proteolytic activity abruptly appears mid-way in embryogenesis and increases steeply during completion of larval differentiation.
Highlights
From the f Laboraton, of Sericultural Science and the I Laboratory of Physical Chemistry, Faculty of Agriculture, Nagoya
Using the purified egg- Such a protease-inhibitorsystem for yolk protein hydrolysis specific protein as a substrate, a protease responsible may not be directly involved in oviparous animals that have for its limited hydrolysis was identified in embryon- heterogeneous yolks
By relatingtheNHz-terminal amino acid se- tively used in embryogenesis (Zhu et al, 1986;Indrasith et al, quences of these peptides to thesequence of the intact 1987).a highly specific protease(s1 may be responsible egg-specific protein, the protease wasshown to cleave for the selective hydrolysis ofegg-specific protein during first at a Lys-Asn site and secondly at Arg-Asp
Summary
Three different assay procedures were used to quantitate thedegradation of egg-specific protein. The standard reaction mixture contained 50 mM sodium phosphate buffer, pH 8.0, 100 pgof egg-specific protein, and enzyme solution (0.1-100pgof protein) in a finalvolume of 300 pl. One unit of enzyme activity in procedure 1 is defined as 1 pg of egg-specificprotein cleaved per min. Assay procedure 2 measured the increase in intensity of an end product which gave a band at 36 kDa on SDS-PAGE. In this case, the 125-kDa intermediate from egg-specific protein instead of the 225-kDa native protein was used as a substrate. The reaction system was identical to thatin procedure 1, and one unit of enzyme activity is defined as 0.25 pg of 36-kDa peptide produced per min. The followingprocedures were used:that of Otto and Bhakdi (1969) for p-nitroanilide derivatives; that of Schwert and Takenaka (1955) forethylestersubstrates; and that of Hummel (1959) for methyl ester substrates
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